Abstract

Chronic exposure to manganese (Mn) produces a neurodegenerative disorder affecting the basal ganglia characterized by reactive gliosis and expression of neuroinflammatory genes including inducible nitric oxide synthase (NOS2). Induction of NOS2 in glial cells causes overproduction of nitric oxide (NO) and injury to neurons that is associated with parkinsonian-like motor deficits. Inflammatory activation of glia is believed to be an early event in Mn neurotoxicity, but specific responses of microglia and astrocytes to Mn during development remain poorly understood. In this study, we investigated the effect of juvenile exposure to Mn on the activation of glia and production of NO in C57Bl/6J mice, postulating that developmental Mn exposure would lead to heightened sensitivity to gliosis and increased expression of NOS2 in adult mice exposed again later in life. Immunohistochemical analysis indicated that Mn exposure caused increased activation of both microglia and astrocytes in the striatum (St), globus pallidus (Gp), and substantia nigra pars reticulata (SNpr) of treated mice compared with controls. More robust activation of microglia was observed in juveniles, whereas astrogliosis was more prominent in adult mice preexposed during development. Co-immunofluorescence studies demonstrated increased expression of NOS2 in glia located in the Gp and SNpr. Additionally, greater increases in the level of 3-nitrotyrosine protein adducts were detected in dopamine- and cAMP-regulated phosphoprotein-32-positive neurons of the St of Mn-treated adult mice preexposed as juveniles. These data indicate that subchronic exposure to Mn during development leads to temporally distinct patterns of glial activation that result in elevated nitrosative stress in distinct populations of basal ganglia neurons.

Differential exposure to Mn in juvenile and adult C57Bl/6J mice induces distinct patterns of astrogliosis in the basal ganglia. Mice were exposed to 0, 10, or 30 mg/kg MnCl2 by daily intragastric gavage as juveniles, adults, or both juveniles and adults. Multiple brain regions in the basal ganglia were assessed for activation of astroglia by immunohistochemical staining for GFAP including the St, Gp, and SNpr. Representative images of the SNpr are depicted for control mice and mice exposed to 30 mg/kg MnCl2 as (A) juveniles, (B) adults, and (C), both juveniles and adults. Scale bar = 10 μm.

Differential exposure to Mn in juvenile and adult C57Bl/6J mice induces distinct patterns of microgliosis in the basal ganglia. Mice were exposed to 0, 10, or 30 mg/kg MnCl2 by daily intragastric gavage as juveniles, adults, or both juveniles and adults. Multiple brain regions in the basal ganglia were assessed for activation of microglia by immunohistochemical staining for Iba-1 including the St, Gp, and SNpr. Representative images of the SNpr are depicted for control mice and mice exposed to 30 mg/kg MnCl2 as (A) juveniles, (B) adults, and (C) both juveniles and adults. Arrowheads indicate cell bodies. Scale bar = 10 μm.

Activated astrocytes in C57Bl/6J mice express NOS2 following differential exposure to Mn as juveniles and adults. Astroglial expression of NOS2 was assessed via co-immunofluorescence in multiple regions of the basal ganglia from mice exposed to Mn. Representative images of the SNpr are presented from control mice and those treated with 30 mg/kg MnCl2 as (A) juveniles, (B) adults, and (C) both juveniles and adults. Images of GFAP and NOS2 expression are shown in green and red channels, respectively, and cell nuclei are highlighted by staining with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) in blue. Yellow areas in merged images indicate colocalization of NOS2 expression in GFAP-positive astrocytes in mice exposed to Mn. Scale bar = 10 μm.

Quantitative analysis of NOS2 expression in GFAP-positive astrocytes reveals regionally and developmentally distinct patterns of astroglial reactivity following exposure to Mn. The percent of GFAP-positive astrocytes expressing NOS2 was determined in mice exposed to 0, 10, or 30 mg/kg MnCl2 by daily intragastric gavage as (A) juveniles, (B) adults, and (C) both juveniles and adults in the St, Gp, and SNpr. Three serial sections were stained for each brain region per hemisphere from an average of three mice per treatment group. For quantitative cell counts, three microscopic fields were evaluated per brain region from each serial section, totaling n = 9 per treatment group. Data represent the mean percentage of GFAP-positive astrocytes expressing NOS2. Different letters denote significant differences between treatment groups, p < 0.05.

Activated microglial cells in C57Bl/6J mice express NOS2 following differential exposure to Mn as juveniles and adults. Microglial expression of NOS2 was assessed via co-immunofluorescence in the St, Gp, and SNpr from mice exposed to Mn. Representative images of the SNpr are presented from control mice and those treated with 30 mg/kg MnCl2 as (A) juveniles, (B) adults, and (C) both juveniles and adults. Images of Iba-1 and NOS2 expression are shown in green and red channels, respectively, and cell nuclei were highlighted by staining with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) in blue. Merged images indicate colocalization of NOS2 expression in Iba-1–positive microglia in mice exposed to Mn. Scale bar = 10 μm.

Quantitative analysis of NOS2 expression in Iba-1–positive microglial cells reveals regionally and developmentally distinct patterns of microglial reactivity following exposure to Mn. The percentage of Iba-1–positive microglia expressing NOS2 was determined in mice exposed to 0, 10, or 30 mg/kg MnCl2 by daily intragastric gavage as (A) juveniles, (B) adults, and (C) both juveniles and adults in the St, Gp, and SNpr. Three serial sections were stained for each brain region per hemisphere from an average of three mice per treatment group. For quantitative cell counts, three microscopic fields were evaluated per brain region from each serial section, totaling n = 9 per treatment group. Data represent the mean percentage of Iba-1–positive microglia expressing NOS2. Different letters denote significant differences between treatment groups, p < 0.05.

Mn exposure increases levels of 3-NTyr protein adducts in basal ganglia neurons. To detect modification of neuronal proteins by peroxynitrite (ONOO−) derived from increased production of NO by activated glia, serial sections from the SNpr of (A) juvenile, (B) adult, and (C) both juvenile and adult mice exposed to 0 and 30 mg/kg MnCl2 were stained with antibodies against the general neuronal marker MAP-2 (green) and 3-NTyr (red) and were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) to identify cell nuclei (blue). Representative images of 3-NTyr–modified proteins indicate colocalization of 3-NTyr adducts with both neuronal soma and dendrites in the SNpr of Mn-treated juvenile mice. Scale bar = 10 μm.

Quantitative analysis of 3-NTyr adducts indicates that modification of neuronal proteins by peroxynitrite correlates with regional patterns of glial activation and NOS2 expression. Specific brain regions were evaluated for colocalization of MAP-2 and 3-NTyr by immunofluorescence in control mice and those exposed to 30 mg/kg MnCl2. Graphs indicate quantification of levels of 3-NTyr protein adducts in neurons from St, Gp, and SNpr of mice exposed as (A) juveniles, (B) adults, and (C) juveniles and adults. Three serial sections were stained for each brain region per hemisphere from an average of three mice per treatment group. For quantitative cell counts, three microscopic fields were evaluated per brain region from each serial section, totaling n = 9 per treatment group. Data indicate increased protein nitration in the neurons of the Gp and SNpr. Different letters denote significant differences between treatments, p < 0.05.

Quantitative analysis of 3-NTyr adducts indicates that modification of specific striatal neuronal proteins by peroxynitrite correlates with regional patterns of glial activation and NOS2 expression. Striatal neurons were evaluated for colocalization of DARPP-32 and 3-NTyr by immunofluorescence in control mice and those exposed to 30 mg/kg MnCl2. Graphs indicate quantification of levels of 3-NTyr protein adducts in neurons from St of mice exposed as (A) juveniles, (B) adults, and (C) juveniles and adults. Three serial sections were stained for each brain region per hemisphere from an average of three mice per treatment group. For quantitative cell counts, three microscopic fields were evaluated per brain region from each serial section, totaling n = 9 per treatment group. Data indicate increased protein nitration in the neurons of the juvenile + adult exposure group. Different letters denote significant differences between treatments, p < 0.05.